The soft lithographic technique is a collection of simple and cost-effective patterning techniques which applies an elastomeric stamp to transfer a nano/micro-scale pattern. Patterning biological materials using soft lithography provides procedurally simple control of the surface chemistry and the cell environments. However, conventional methods for generating microstructures on a substrate require expensive clean room facilities and skillful training. Here we report a simple and inexpensive clean-room free process using a conventional photomask film as a master to fabricate elastomeric stamps or microfluidic channels. This ultra rapid prototyping technique was applied to print FITC labeled poly-L-lysine with a 10 µm feature size on a glass substrate using soft lithographic processes, such as micro-contact printing and micromolding in capillaries, for patterning human hepatocellular carcinoma cells, human skin fibroblasts and hippocampal neurons from E-18 Sprague-Dawley rat. This novel technique using a photomask film as a master would be very useful ‘hands-on’ tool for the generation of micro-patterned chemical or biological assays using cells and proteins.